DISCUSSION 97

We predicted that USVs at different ages would be differentially disrupted following PCE and that differences would correlate with variations in development of specific brain regions. As hypothesized, cocaine-induced changes in neonatal vocalizations were sex and age-dependent. On PND 1, CC decreased the number of vocalizations in both sexes and CC males had a smaller percentage of calls with at least one observable harmonic than did UN males at this age. Following a painful experience, human infants emit cries with fewer harmonics which are then perceived to be more urgent (Porter et al., 1986). Maternal substance abuse is correlated with neglect in humans (Leventhal et al., 1997) and rodents (Nelson et al., 1998;Johns et al., 1994) perhaps partially mediated through variation in infant cues (Johns et al., 2005). Vocalization characteristics, including harmonics, could potentially be an important cue impacting human and rodent early care. For instance, if human male infants prenatally exposed to cocaine emit expirations that have acoustic differences in harmonics this could influence maternal perception (i.e. feeling of urgency) and hence parental response toward the infant. No study to date has explored the impact of harmonics on early dam-pup interactions in a rodent model. Since gestational cocaine-exposure has been found to alter maternal behavior (with the greatest deficits occurring around PPD one) future studies should target early translational behavioral differences in CC pups and their impact on the maternal environment to continue elucidating the mechanisms underlying neglect.

The PAG is a region critical for triggering vocalizations (Jurgens, 2009;Larson and Kistler, 1984) in response to limbic emotional (Adamec et al., 2003;Zhao et al., 2009) and somatosensory input (Mayer, 1984;Sandkuhler et al., 1991). We hypothesized that variation in PAG development would therefore impact vocalization production, even during the neonatal period when vocalizations are speculated to be an acoustic by-product of thermoregulation (Blumberg and Alberts, 1990). Vocalization changes in CC offspring did not however correlate with altered PAG development on PND 1. Future studies targeting later proliferation times (i.e. GDs 16-17) might help to clarify

findings as proliferation in the PAG occurs between GDs 13-17 in a ventral to dorsal gradient (Altman and Bayer, 1981). Since neonatal vocalizations are thought to be produced via a ‘brainstem model’ in that regions rostral to the midbrain are not required for vocalization production during early infant development (Newman, 2007), perhaps other regional changes in the brainstem such as the nucleus ambiguous (Wetzel et al., 1980), should be explored in CC offspring.

PND 14 was found to be a peak vocalizing period for all offspring, as evidence in more vocalizations that were longer and had shorter intervals between individual USVs than at other ages. PND 14 in rodents is thought to be roughly equivalent in age to one month old human infants who have a peak crying period around six to eight weeks (Barr et al., 1996;Prescott, 1975) followed by a decrease in vocalizations (Barr et al., 1996;Prescott, 1975). Rodent infants show a similar developmental shift to human infants, first producing high rates of isolation-induced USVs (Hofer et al., 1998;Hofer and Shair, 1980;Shair et al., 2003) followed by signs of fear-like behavior (i.e. freezing and decreasing USV production) when in an aversive environment (i.e. predator odor or isolation) (Kabitzke and Wiedenmayer, 2011). Our age comparisons support this developmental progression in rodent infants with both CC and CS-exposure in males prolonging higher rates of vocalizing. While human PCE has been suggested to decrease neonatal crying (Corwin et al., 1992) similar to what we observed on PND 1, it has also been found to increase excessive crying and irritability (Keller, Jr. and Snyder-Keller, 2000;Eyler et al., 1998). In general, “excessing crying” has been referred to as a common symptom following substance exposure (Galanter and Kleber, 2008). Since prenatal stress has also been shown to result in excessive crying (van der Wal et al., 2007) with a peak period of fussiness occurring between three and six months of age (Wurmser et al., 2006), similar findings in both CC and CS males could suggest effects at this age may reflect a common or similar early developmental stress effect. As not all differences were identical for CC and CS-exposed males, PCE specific changes are likely resulting from more direct effects of drug exposure. The sex specific effects suggest that males may be more sensitive to these manipulations which support previous reports. Sex-dependent developmental differences in rats have been reported for a number of

measures following PCE (Johns et al., 2002;Lewis et al., 2009;Hamilton et al., 2011;Dow-Edwards, 2010) and prenatal stress (Bale, 2011) with males suggested to be more vulnerable to prenatal complications, including stress (Gerardin et al., 2011). These findings are particularly interesting in light of the many neurodevelopmental disorders (many with increased prevalence in males) that are associated with altered early communication in humans. For instance, parents of children with autism spectrum disorder often report that they cannot understand why their child is crying in the first year. These parents describe their child’s cries as unexpected and loud (Esposito and Venuti, 2010). Altered crying behavior in males could also have a detrimental impact on the maternal environment, especially in populations more vulnerable to neglect-like behavior such as those abusing cocaine, further strengthening the need for more research exploring the implications of sex-dependent changes in vocalizations.

We hypothesized that developmental delays in fear-related circuitry would correlate with altered vocalizations on PND 21 in CC offspring, including increased rates of vocalizations and delayed onset of 22 kHz USVs. As the limbic system matures in the developing human infant the first displays of fear-like behavior begin to emerge (Herschkowitz et al., 1997). Previous animal studies indicate that fear-like behavior is associated with plasticity of the VMH (Pagani and Rosen, 2009) and amygdala (Collins, 2011;Takahashi et al., 2007). In the present study CC and CS female (significant) and male (non-significant) offspring had a reduction in the percentage of USVs that fell into the 22 kHz range on PND 21 suggesting that CC and CS exposure may alter emergence of USV expression associated with fear-like behavior in both sexes though this effect was stronger in females in the present study.

On PND 21 CC and CS males had a greater density of VMH neurons compared to controls. Increased neuronal density (and larger percentage of BrdU cells co-labeled as a neuron) was found to positively correlate with the total duration of vocalizations suggesting dysregulated VMH development (previously suggested as playing a role in infant crying (Joseph, 1996)), may possibly have contributed to the altered crying in CC and CS males. Greater neuron density in the VMH and

CeA was negatively correlated with the percentage of 22 kHz USVs emitted, suggesting a role for CeA and VMH developmental differences with delayed onset of fear-like behavior which should be further explored. It is important to note that data appears to be bi-modal for many acoustic measures. Fear circuitry has recently been shown to be modulated by the medial prefrontal cortex (Chan et al., 2011) and hence other regions could be playing a role in vocalization production and control as offspring appear to fall into two categories: those that vocalize and those that don’t suggesting other mechanisms of inhibition are occurring.

We were surprised that CC-exposure in males was associated with greater neuron density in the VMH on PND 21 as we hypothesized that they would have delayed neuronal maturation. PCE has previously been shown to decrease proliferation but not effect cell survival (Lee et al., 2008), increase apoptosis in the fetal brain (Xiao and Zhang, 2008), delay postnatal astroglial maturation (Clarke et al., 1996), disrupt migration, and/or enhance differentiation in offspring (Lee et al., 2011). Few studies have examined later periods in development for maturational changes in the brain. Based on these results, increased differentiation could actually be a mechanism to explore, however, it is important to note that cell death continues to occur in the brain through adolescence and is also sexually dimorphic (Nunez et al., 2001;Nunez et al., 2002) thus future studies are needed to clarify these findings.

CC and CS males vocalized at higher amplitudes on PND 21 compared to UN males and regional differences in the VMH were related to amplitude of the loudest call emitted during testing. Stimulation of the VMH has been previously associated with increased USV amplitude parallel to pain-induced increases in USV amplitude (Borszcz, 2006). It was interesting that the correlation of VMH with amplitude was age-dependent. PND 14 offspring showed a negative inverse relationship between amplitude and the percentage of BrdU cells that co-labeled as a neuron (Figure 19P) while PND 21 offspring showed a positive relationship between amplitude and the density of VMH neurons (Figure 20P). Defensive behavior and what is perceived as a threatening stimulus changes in an age- dependent manner during early development (Wiedenmayer, 2009). Differential age-specific

relationship between VMH development and amplitude of calling could represent a developmental change in VMH function or changing sensitivity to the isolation/ cold scale stimulus used for USV elicitation in this study.

Recent findings suggest changes in the amplitude of vocalizations are associated with early differences in the rearing environment (i.e. maternal care), and might be functionally relevant as a behavioral marker of early environmental differences (Wohr et al., 2008a). With this in mind, it is important to note that a limitation of this study is that we did not differentiate between the consequences of prenatal cocaine-induced changes in offspring vocalizations with that of differences in the postnatal environment. Previous studies have found that early environmental experience alters vocalizations in rodent pups. Specifically, pups experiencing five days of brief maternal separation/deprivation (an animal model of maternal neglect) from PND two through six vocalized less and show subtle changes in the sonographic structure of USVs than control pups when re-isolated from their dam and litter on PNDs seven and twelve (Zimmerberg et al., 2003b;Zimmerberg et al., 2003a). Maternal deprivation has been shown to decrease neurogenesis in the hippocampus (Fabricius et al., 2008) and to effect neural proliferation differentially in male and female offspring. These gender differences are speculated to coincide with behavioral differences in adulthood and imply that early life stress establishes sex differences in neural plasticity, contributing to alterations in the HPA axis (Oomen et al., 2009). Prenatal stress also differentially affects neural proliferation in male and female offspring (Weinstock, 2007;Vaido et al., 2000;Mandyam et al., 2008). These studies and our findings here might suggest differences in vocalizations and differences observed in the VMH on PND 21 might be primarily related to environmental-induced differences. It is interesting that only males and not females showed an increase in both amplitude and total duration of USVs on PND 21 which coincided with increased neuronal density in the VMH. Future studies employing cross fostering paradigms should focus on determining the role of the postpartum environment as an independent mediator of these effects.

As hypothesized, we did not observe any correlations between offspring NAc development and variations in vocalizations. Based on the testing paradigm employed in the present study (eliciting vocalizations via cold isolation) we hypothesized that developmental differences in fear- associated regions would correlate with differences in USVs and not reward-associated regions. The differences observed in the NAc core of CC offspring are interesting and could impact infant social behavior, including vocalizations, in positive/ rewarding testing paradigms (i.e. play behavior or social interactions with dam). Just like rodent dams find pups highly salient the presence of the dam is thought to also be very rewarding to the pup. As early as PND 11, pups undergo “contact quieting”, in which they attenuate USV production, following reunion with dam. Contact quieting was found to be dopamine-dependent, involve the NAc, and dam-specific as reunion with littermates is was not salient enough to attenuate USVs (Shair et al., 2009). Additionally, re-isolation following dam reunion leads to maternal potentiation, a greater number of calls observed than what was previous observed in isolation before dam reunion, however re-isolation following litter mate reunion does not (Hofer et al., 1998;Hofer et al., 1994;Muller et al., 2009). These studies and studies showing anxiolytic drugs can reduce isolation-induced USVs (Hamed et al., 2009;Winslow and Insel, 1991) underlies theories that dam-reunion attenuated USVs decreases the stress of the pup through rewarding salience of the dam. Developmental differences observed in the NAc core of CC offspring could have profound impact on dopamine signaling and hence the rewarding value of the dam to the offspring. Human studies have suggested that cocaine-exposed mothers have a more difficult time soothing their infant (Eiden et al., 2009a;Eiden RD et al., 2011); which could be a consequence of altered infant NAc development. Most studies examining USVs using an isolation testing paradigm. In light of the present findings, other testing paradigms (i.e. contact quieting, social play) should be explored as differences observed in these paradigms may be more related to mother-infant interactions.